PROJECT SUMMARY The serrated pathway to colorectal carcinoma (CRC) accounts for 35% of all CRC. A subset of lesions arising along this pathway are characterized by methylation of CPG islands in the promoters of genes involved in tumor suppression and mismatch repair leading to their epigenetic silencing. Accordingly, this group of tumors is known by the acronym CIMP (CpG island methylator phenotype). Epidemiological studies suggest that serrated lesions leading to CIMP CRCs may be more likely to be missed with colonoscopy perhaps due to their predominance in the proximal colon, morphology, or rapid growth rate. CIMP tumors have also been associated with an increased risk of CRC-related death. Despite the public health importance, the underlying cause of these aberrant epigenetic changes remains unknown. Identification of factors contributing to aberrant methylation in CIMP could lead to new strategies for risk assessment and preventative therapeutic interventions. We recently conducted a preliminary study to examine the role of the microbiome in CIMP CRCs. Our research suggests that prominent butyrate-producing bacterial (BPB) populations and the butyrate they produce are deficient in CIMP compared to Non-CIMP CRCs. We hypothesize that deficiency in colonic butyrate, a bacterial metabolite of fiber fermentation with known anti-neoplastic and epigenetic effects, is the primary cause of abnormal CpG island methylation in CIMP CRCs. To test this hypothesis, we will characterize biopsies from patients with CIMP and Non-CIMP tumors and pursue mechanistic studies in a transgenic mouse model. In Aim 1, we will use high-throughput sequencing of the bacterial 16S rRNA gene, to evaluate group-specific differences in the microbiota between patients with CIMP and Non-CIMP tumors and investigate functional differences between the CIMP and Non-CIMP tissues, including synthesis of butyrate and other short-chain fatty acids, such as propionate and acetate. In Aim 2, we will quantitate and compare concentrations of butyrate and other fatty acids directly in tumors and normal tissue from patients with CIMP and Non-CIMP tumors. In Aim 3, we will interrogate the transgenic HBUS mouse model of serrated tumorigenesis to investigate the functional consequences of dietary fiber deficiency and deficient BPB populations and butyrate on tumor progression and CIMP. At the conclusion of these studies, we will have expanded our knowledge of the role of the microbiota in different molecular phenotypes of CRC and provided evidence for or against a causal role for BPB and butyrate deficiency in aberrant CpG island methylation found in CIMP tumors.